Expression unit of pet hydrolase, recombinant vector, recombinant pseudomonas putida, and construction method therefor and use thereof

Abstract

The present invention relates to an expression unit of a PET hydrolase, a recombinant vector, recombinant Pseudomonas putida, and a construction method therefor and the use thereof. The expression unit of PET hydrolase comprises a constitutive expression promoter of Pseudomonas putida, an anchor protein gene, a PET hydrolase gene and a protein expression terminator of Pseudomonas putida, all of which are linked. In the above expression unit of PET hydrolase, the anchor protein gene is linked to the PET hydrolase gene, and the promoter and terminator of Pseudomonas putida are linked thereto, which enables the display of PET hydrolase on the surface of Pseudomonas putida. The resulting PET hydrolase has a relatively high catalytic activity and can be used for the enzymatic degradation of PET plastics. Experiments verify that recombinant Pseudomonas putida P. putida / pMCS-PETase-AT containing the above expression unit exhibits the catalytic enzyme activity of PET hydrolase on BHET, with a higher catalytic activity compared to a wild-type strain.

Description

PET hydrolase expression unit, recombinant vector, recombinant Pseudomonas putida and construction method and application thereof Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to an expression unit of PET hydrolase, a recombinant vector, a recombinant Pseudomonas putida and a construction method and application thereof. Background Art

[0002] PET is a condensation polymer of terephthalic acid (PTA) and ethylene glycol (EG). Plastic products primarily based on PET account for 18% of the global plastics market, with an annual production of 33 million tons. Due to its excellent physical and chemical properties, PET is widely used in packaging, containers, and clothing. However, due to its high molecular weight, high hydrophobicity, and high chemical bond energy, synthetic plastics are unable to penetrate biofilms and are difficult to degrade by microorganisms. This ultimately leads to their long-term persistence and accumulation in the environment, making "white pollution" a global problem. Currently, conventional treatment options for waste PET plastics include recycling (14%), incineration (14%), and landfill (72%). These methods are relatively simple, but they have limitations such as high costs, numerous byproducts, and secondary pollution. In recent years, researchers have gradually developed new technologies for recycling PET plastics, such as thermomechanical recycling and chemical recycling. However, for PET that does not meet recycling standards (such as low viscosity or high dye content) and PET waste from the textile industry, no suitable treatment technologies have been explored to date.

[0003] In recent years, the biodegradation of PET plastics has emerged as a potential new, environmentally friendly strategy, transforming discarded PET plastics into biomass through biological metabolic processes and integrating them into the geochemical cycle. The scientific community has long sought effective PET biodegradation methods, primarily through the exploration of microbial resources and related enzymatic research. Currently, discovered enzyme resources with PET depolymerization capabilities include cutinases, lipases, and esterases from species such as Thermobifida, Saccharomonospora viridis, Ideonella sakaiensis, Humicola insolens, and Fusarium oxysporum. Among them, Ideonella sakaiensis 201-F6 is a microorganism reported to utilize PET as its primary carbon source for growth and metabolism. A PET hydrolase (PETase) with PET depolymerization capabilities has been discovered from this bacterium. This PETase can decompose PET into terephthalic acid (TPA), ethylene glycol (EG), and bis(2-hydroxyethyl) terephthalate (BHET). The enzyme's greatest characteristic is its ability to efficiently catalyze reactions under mild conditions (20-40°C). Its PET hydrolysis efficiency at room temperature is higher than that of previously reported cutinases, lipases, and esterases. However, the strain was unable to grow when PET was its sole carbon source, indicating that the PET hydrolase expressed by the strain was inefficient in decomposing PET, possibly related to the amount of PET hydrolase secreted extracellularly. Due to the lack of genetic manipulation tools for genetically modifying the bacterial genome, further metabolic engineering studies on this strain have been difficult.

[0004] Currently, conventional methods for processing PET plastics mainly include physical methods, chemical methods, incineration, and landfill. The physical method decomposes the recycled PET plastic by sorting, grinding, heating, and other processes. This method cannot completely degrade the plastic and is prone to generating new environmental pollutants (microplastics). The chemical method degrades PET plastic through processes such as glycolysis, alcoholysis, hydrolysis, and aminolysis. These processes usually require high temperatures and the addition of catalysts, are costly, and are prone to secondary pollution. Incineration can emit toxic and harmful substances, polluting water and air. Landfill treatment takes a long time and can easily cause soil compaction, hindering crop growth.

[0005] Currently, research on the biodegradation of PET plastics primarily focuses on the discovery, development, and utilization of PET-degrading enzymes and microorganisms. While enzymatic degradation of PET plastics has achieved some progress, this method is only suitable for processing the recycled portion (14%) of PET plastics and requires a relatively high reaction temperature (72°C). In situ treatment of waste PET plastics that accumulate in the environment is difficult, limiting its application in natural environments. Microorganisms, as natural decomposers, thrive in diverse and diverse environments and possess highly evolved and adaptable abilities. Degrading plastics through microbial means is environmentally friendly and energy-saving, making it a safe and ecologically sound degradation method. Currently, the production of PET hydrolases primarily relies on intracellular expression in genetically engineered Escherichia coli, which not only requires fragmentation but also presents numerous challenges in downstream separation and purification. PET plastics cannot penetrate the cell membrane to enter the cell for degradation, requiring the PET hydrolase to be secreted extracellularly. Furthermore, the secretion of PET hydrolases can irreversibly damage the integrity of the cell membrane, impacting cell growth. Therefore, a suitable host is sought to express PET hydrolase and to immobilize the enzyme on the surface of the cell membrane, thereby reducing the impact of the secretion and expression of PET hydrolase on cell growth and improving its degradation efficiency of PET plastics.

[0006] Summary of the Invention

[0007] Based on this, it is necessary for the present application to provide an expression unit of PET hydrolase, which can be used to display PET hydrolase on the cell surface. The obtained PET hydrolase has high catalytic activity and can be used for enzymatic degradation of PET plastics.

[0008] In addition, it is also necessary to provide a recombinant vector, a recombinant Pseudomonas putida, and a construction method and application thereof.

[0009] A PET hydrolase expression unit comprises a connected Pseudomonas putida constitutive expression promoter, an anchor protein gene, a PET hydrolase gene and a Pseudomonas putida protein expression terminator.

[0010] In the aforementioned PET hydrolase expression unit, the anchor protein gene is linked to the PET hydrolase gene and connected to the Pseudomonas putida promoter and terminator, enabling the PET hydrolase to be displayed on the surface of Pseudomonas putida. The resulting PET hydrolase has high catalytic activity and can be used for enzymatic degradation of PET plastics. Experimental verification has shown that the recombinant Pseudomonas putida / pMCS-PETase-AT containing this expression unit exhibits catalytic activity against BHET, with higher catalytic activity than the wild-type strain.

[0011] In one embodiment, the sequence of the PET hydrolase gene is shown as SEQ ID No. 1.

[0012] In one embodiment, the sequence of the anchoring protein gene is shown as SEQ ID No. 2 or SEQ ID No. 3.

[0013] In one embodiment, the nucleotide sequence of the expression unit is shown as SEQ ID No. 4 or SEQ ID No. 5.

[0014] A recombinant vector comprising the above-mentioned expression unit.

[0015] In one embodiment, the original vector corresponding to the recombinant vector is selected from one of the pBBR1MCS vector, the pSEVA331 vector, the pYMB03 vector and the pJB866 vector.

[0016] A recombinant Pseudomonas putida, comprising the above-mentioned expression unit; or, comprising the above-mentioned recombinant vector.

[0017] In one embodiment, the starting host bacteria of the recombinant Pseudomonas putida is selected from one of Pseudomonas putida KT2440, Pseudomonas putida NBRC 14164, Pseudomonas putida S16, and Pseudomonas putida PP1;

[0018] Furthermore, the starting host bacteria of the recombinant Pseudomonas putida is Pseudomonas putida PP1 bacteria with CCTCC NO: M 20231659.

[0019] A method for constructing a recombinant Pseudomonas putida comprises the following steps:

[0020] An expression unit for synthesizing PET hydrolase, the expression unit comprising a connected Pseudomonas putida constitutive expression promoter, an anchor protein gene, a PET hydrolase gene, and a Pseudomonas putida protein expression terminator;

[0021] connecting the expression unit to a Pseudomonas putida expression vector to obtain a recombinant vector;

[0022] The recombinant vector is transformed into a host Pseudomonas putida to obtain a recombinant Pseudomonas putida.

[0023] The use of the above expression unit, or the above recombinant vector, or the above recombinant Pseudomonas putida, or the recombinant Pseudomonas putida obtained by the above construction method of recombinant Pseudomonas putida in degrading PET plastic.

[0024] A PET degrading agent comprises: the recombinant Pseudomonas putida or the recombinant Pseudomonas putida obtained by the construction method of the recombinant Pseudomonas putida.

[0025] A PET degradation method comprises the following steps: mixing cells with PET plastic and reacting the cells to degrade the PET plastic, wherein the cells are the recombinant Pseudomonas putida or the recombinant Pseudomonas putida obtained by the construction method of the recombinant Pseudomonas putida. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 is a comparison of the PET degradation capabilities of the recombinant Pseudomonas putida constructed in Example 1 and Example 2. DETAILED DESCRIPTION

[0027] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, the following detailed description of the specific embodiments of the present invention is provided in conjunction with specific embodiments and accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0028] One embodiment of the present application provides an expression unit for PET hydrolase, which includes a connected Pseudomonas putida constitutive expression promoter, an anchor protein gene, a PET hydrolase gene, and a Pseudomonas putida protein expression terminator.

[0029] Because secretory expression of the PET hydrolase sequence carries a secretory peptide at its N-terminus, which can cause cell membrane lysis, cell surface display technology is required to display it on the cell membrane to improve the enzyme's PET degradation efficiency. Compared to commonly used engineered host bacteria such as Escherichia coli or Pichia pastoris, Pseudomonas putida not only has advantages such as broad environmental adaptability, the ability to synthesize excess intracellular energy storage materials, and a mature genetic operating system, but also can further degrade terephthalic acid and ethylene glycol, the products of PET hydrolysis, reducing the problem of secondary environmental pollution caused by degradation products.

[0030] In one embodiment, the sequence of the PET hydrolase gene is shown in SEQ ID No. 1. The PET hydrolase is derived from Ideonella sakaiensis 201-F6 and has the advantage of having the highest catalytic activity towards PET at a relatively low temperature (30° C. to 40° C.).

[0031] In one embodiment, the anchoring protein gene is inaK-N or estP-C. Specifically, the sequence of the anchoring protein gene is shown in SEQ ID No. 2 or SEQ ID No. 3. Using the above two anchoring protein genes has the advantages of high efficiency and stable performance of surface display of PET hydrolase.

[0032] In one embodiment, the expression unit is constructed in the order of Pseudomonas putida constitutive expression promoter + anchor protein gene + PET hydrolase gene + Pseudomonas putida protein expression terminator from 5' to 3' direction. Specifically, the nucleotide sequence of the PET hydrolase expression unit is shown in SEQ ID No. 4 or SEQ ID No. 5.

[0033] In the above-mentioned PET hydrolase expression unit, the anchor protein gene is connected to the PET hydrolase gene, and is also connected to the promoter and terminator of Pseudomonas putida, so that the PET hydrolase can be displayed on the surface of Pseudomonas putida. The obtained PET hydrolase has high catalytic activity and can be used for enzymatic degradation of PET plastics.

[0034] One embodiment of the present application further provides a recombinant vector comprising the above-mentioned expression unit.

[0035] The above-mentioned recombinant vector is used to clone or express PET hydrolase and can be used for enzymatic degradation of PET plastics.

[0036] The original vector corresponding to the recombinant vector is selected from one of the pBBR1MCS vector, the pSEVA331 vector, the pYMB03 vector, and the pJB866 vector. The use of the above original vectors is conducive to the surface display of PET hydrolase on Pseudomonas putida.

[0037] One embodiment of the present application further provides a recombinant Pseudomonas putida, which contains the above-mentioned expression unit; or the above-mentioned recombinant vector.

[0038] The starting host strain for the recombinant Pseudomonas putida is selected from Pseudomonas putida KT2440, Pseudomonas putida NBRC 14164, Pseudomonas putida S16, and Pseudomonas putida PP1. These starting hosts facilitate surface display of the PET hydrolase, resulting in high biocatalytic activity. This enables the application of enzymatic degradation of PET plastics, and the enzyme does not require isolation or purification, making preparation and use simple.

[0039] Furthermore, the starting host bacteria of the recombinant Pseudomonas putida is Pseudomonas putida PP1 bacteria with CCTCC NO: M 20231659. Pseudomonas putida PP1 bacteria were deposited in the China Center for Type Culture Collection (CCTCC) on September 11, 2023, with the deposit number CCTCC NO: M 20231659.

[0040] The above-mentioned recombinant Pseudomonas putida can display PET hydrolase on its surface, and the bacteria can directly react with the substrate as a whole-cell catalyst without the need to break the cells and purify the PET hydrolase (PETase), making the biological degradation of PET plastics more convenient and efficient, with high biocatalytic activity, realizing the application of enzymatic degradation of PET plastics, and the enzyme does not need to be separated and purified, and is simple to prepare and use.

[0041] Furthermore, the surface-displayed enzyme is easy to recycle, has high stability, and is reusable. The surface-displayed enzyme can more fully and effectively contact with the rigid substrate such as PET to maximize the catalysis of the substrate.

[0042] One embodiment of the present application further provides a method for constructing a recombinant Pseudomonas putida, comprising the following steps S110-S130:

[0043] S110. Synthesize the expression unit of the PET hydrolase, wherein the expression unit comprises a connected Pseudomonas putida constitutive expression promoter, an anchor protein gene, a PET hydrolase gene, and a Pseudomonas putida protein expression terminator.

[0044] The detailed description of the expression unit of PET hydrolase is given above and will not be repeated here.

[0045] S120. Connect the expression unit to a Pseudomonas putida expression vector to obtain a recombinant vector.

[0046] The Pseudomonas putida expression vector is selected from one of the pBBR1MCS vector, the pSEVA331 vector, the pYMB03 vector, and the pJB866 vector. The use of the above vectors is conducive to the surface display of PET hydrolase on Pseudomonas putida.

[0047] Furthermore, the starting host bacteria of the recombinant Pseudomonas putida is Pseudomonas putida PP1 bacteria with CCTCC NO: M 20231659. Pseudomonas putida PP1 bacteria were deposited in the China Center for Type Culture Collection (CCTCC) on September 11, 2023, with the deposit number CCTCC NO: M 20231659.

[0048] Specifically, the expression unit is connected to the Pseudomonas putida expression vector using seamless cloning technology to obtain a recombinant vector.

[0049] S130. Transform the recombinant vector into the host Pseudomonas putida to obtain recombinant Pseudomonas putida.

[0050] The host Pseudomonas putida is selected from one of Pseudomonas putida KT2440, Pseudomonas putida NBRC 14164, Pseudomonas putida S16, and Pseudomonas putida PP1. These host Pseudomonas putida facilitate the surface display of a PET hydrolase, exhibit high biocatalytic activity, and enable the application of enzymatic degradation of PET plastics. Furthermore, the enzyme does not require isolation or purification, making preparation and use simple.

[0051] Furthermore, the starting host bacteria of the recombinant Pseudomonas putida is Pseudomonas putida PP1 bacteria with CCTCC NO: M 20231659. Pseudomonas putida PP1 bacteria were deposited in the China Center for Type Culture Collection (CCTCC) on September 11, 2023, with the deposit number CCTCC NO: M 20231659.

[0052] The recombinant Pseudomonas putida obtained by the above construction method can display PET hydrolase on its surface and has high biocatalytic activity, thus realizing the application of enzymatic degradation of PET plastics. Moreover, the enzyme does not need to be separated and purified, and is simple to prepare and use.

[0053] Furthermore, the surface-displayed enzyme is easy to recycle, has high stability, and is reusable. The surface-displayed enzyme can more fully and effectively contact with the rigid substrate such as PET to maximize the catalysis of the substrate.

[0054] This study also provides a PET degrader, comprising: the above-mentioned recombinant Pseudomonas putida, or the recombinant Pseudomonas putida obtained by the above-mentioned construction method of recombinant Pseudomonas putida.

[0055] The above-mentioned PET degradation agent contains surface-displayed PET hydrolase, has high BHET catalytic enzyme activity, and has high degradation efficiency for PET plastics.

[0056] It is understood that the PET degrading agent may contain only the above-mentioned recombinant Pseudomonas putida, or may contain other components in addition to the above-mentioned recombinant Pseudomonas putida, such as auxiliary materials.

[0057] This study provides a PET degradation method, comprising the following steps: mixing cells with PET plastic and reacting them to degrade the PET plastic, wherein the cells are the above-mentioned recombinant Pseudomonas putida or the recombinant Pseudomonas putida obtained by the above-mentioned construction method of recombinant Pseudomonas putida.

[0058] In the above-mentioned PET degradation method, the recombinant Pseudomonas putida that displays PET hydrolase on its surface can be directly used as a whole-cell catalyst to react with the substrate, without the need to disrupt the cells and purify the PET hydrolase (PETase), making the biological degradation of PET plastic more convenient and efficient.

[0059] Specifically, the step of mixing the cells with the PET plastic and reacting the mixture includes: using a pH 9 buffer solution to prepare the recombinant Pseudomonas putida into a bacterial suspension, and adding the suspension into the PET plastic for reaction.

[0060] Furthermore, before the step of mixing the cells with the PET plastic and reacting them, the method further includes pre-treating the PET plastic: rinsing the PET plastic with Triton X 100 at 30°C, then washing the PET plastic with water, and then rinsing the PET plastic with a Na2CO3 solution at 30°C, washing the PET plastic with water, and drying the PET plastic to obtain a pre-treated PET plastic. This pre-treatment can remove impurities on the surface of the PET plastic, preventing them from affecting the results of the PET hydrolase-catalyzed reaction.

[0061] The following are specific examples.

[0062] Unless otherwise specified, the drugs and instruments used in the examples are all conventionally selected in the art. Experimental methods without specific conditions specified in the examples are generally carried out under conventional conditions, such as those described in literature or books, or methods recommended by the kit manufacturer.

[0063] In the following examples, Pseudomonas putida PP1 was deposited in the China Center for Type Culture Collection (CCTCC) on September 11, 2023, with the accession number CCTCC NO: M 20231659.

[0064] Example 1:

[0065] Construction and application of recombinant Pseudomonas putida displaying PET hydrolase on its cell surface (P. putida / pMCS-INP-PETase)

[0066] Specifically, the core expression unit of the cell surface display PET hydrolase (PETase) DNA recombinant vector is constructed in the following order from 5' to 3' direction: a strong constitutive expression promoter for Pseudomonas putida (Pp_Pgro4) + an anchoring protein gene (inaK-N, codon-optimized for Pseudomonas putida) + a PET hydrolase ORF (PETaseIs, codon-optimized for Pseudomonas putida) + a Pseudomonas putida protein expression terminator (Pp_T0). The specific gene sequence is shown in SEQ ID No. 4, wherein: the Pp_Pgro4 nucleotide sequence is shown in SEQ ID No. 4, positions 1 to 244; the inaK-N nucleotide sequence is shown in SEQ ID No. 2; the PETaseIs nucleotide sequence is shown in SEQ ID No. 1; and the Pp_T0 nucleotide sequence is shown in SEQ ID No. 4, positions 1568 to 1676.

[0067] Furthermore, through gene synthesis, we commissioned Sangon Biotech (Shanghai) Co., Ltd. to synthesize the cell surface-displayed PET hydrolase expression unit in pMCS-INP-PETase (with Xbal and SpeI restriction sites added upstream and downstream, respectively).

[0068] Further, the pBBR1MCS plasmid (purchased from MiaoLingBio) was extracted and double-digested with Xbal and SpeI to recover the vector backbone fragment. Based on seamless cloning technology, the cell surface display PET hydrolase expression unit (as shown in SEQ ID No. 4) was cloned into the pBBR1MCS linear vector after enzyme digestion to obtain the recombinant DNA plasmid pMCS-INP-PETase (pBBR1MCS + Pp_Pgro4 + inaK-N + PETaseIs + Pp_T0), which was then transferred into Escherichia coli DH5α. After kanamycin resistance screening, colony PCR verification, and sequencing verification, the correct recombinant plasmid was obtained.

[0069] Furthermore, the pMCS-INP-PETase recombinant DNA plasmid was introduced into the host bacteria Pseudomonas putida PP1 to obtain the recombinant Pseudomonas putida P. putida / pMCS-INP-PETase displaying the PET hydrolase on the cell surface of the present invention.

[0070] Furthermore, the specific method of the above-mentioned electric shock conversion is as follows:

[0071] 1) Inoculate a single clone of Pseudomonas putida PP1 activated on an LB plate into a 4 mL / 12 mL shake tube and incubate at 30°C for 12-15 hours.

[0072] 2) Collect cells by centrifugation in a 2 mL EP tube at 13,000 × g for 2 min at room temperature;

[0073] 3) Add 1 mL of 300 mM sucrose solution to a 2 mL EP tube, resuspend the cells twice, and centrifuge at 13,000 × g for 2 minutes at room temperature to collect the cells;

[0074] 4) Resuspend the cells in 100 μL of 300 mM sucrose solution to obtain competent cells;

[0075] 5) Mix 500 ng of recombinant plasmid pMCS-SP1-PETase with 100 μL of competent cells and let stand for 5 minutes;

[0076] 6) Add the above mixture to a sterile electroporation cuvette (0.2 cm), electroporate once at 2500 V, 25 μV, and 200 Ω, add 1 mL of LB medium, transfer the cells to a 12 mL shake tube, and resuscitate at 30°C for 2 h.

[0077] 7) Spread 100 μL of the above recovery culture medium on an LB plate containing kanamycin (50 mg / L) and incubate at 30°C for 12 h.

[0078] Furthermore, a single colony of recombinant P. putida / pMCS-INP-PETase displaying PET hydrolase on its cell surface was picked from an LB plate containing kanamycin (50 mg / L) and inoculated into 50 mL / 250 mL seed medium. The culture was incubated at 30°C, 200 rpm for 12 h. The seed solution was centrifuged, and the fermentation medium was inoculated to a final seed concentration of 0.1 at OD600. The culture was incubated at 30°C, 200 rpm for 48 h. The bacterial solution was collected and the cells were centrifuged at 12,000 rpm for subsequent whole-cell catalytic reactions using the recombinant strain.

[0079] Seed culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, solvent is deionized water, pH value is natural.

[0080] Fermentation medium: glycerol 5 g / L, ammonium chloride 1 g / L, KH2PO4 3 g / L, MgSO4 2 mM, Na2HPO4·12H2O 17.1 g / L, NaCl 0.5 g / L, yeast extract 2 g / L, CaCl2 0.5 g / L, VB1 40 mg / L, 1 mL / L trace element solution, solvent is deionized water, pH value is natural; trace element solution composition: ZnSO4·7H2O 2 g / L, MnCl2·2H2O 1.0 g / L, Na2B4O7 0.3 g / L, Na2MoO4·2H2O 0.2 g / L, CuSO4·7H2O, 0.2 g / L, CoCl2·6H2O 0.4 g / L, solvent is deionized water.

[0081] Whole-cell catalytic reaction of recombinant Pseudomonas putida / pMCS-INP-PETase

[0082] PET film treatment: Cut the PET film into 0.2 cm × 0.5 cm strips, rinse several times with double-distilled water, add Triton X 100 and heat-wash 1-2 times, wash with double-distilled water to remove Triton X 100 residue until no residue remains, add Na2CO3 solution and heat-wash several times, rinse several times with double-distilled water, and dry in a clean Petri dish at 60°C until used.

[0083] Take an appropriate amount of the recombinant P. putida / pMCS-INP-PETase fermentation broth, centrifuge at 3500 rpm for 5 min, add Glycine / NaOH pH = 9 buffer to resuspend, centrifuge at 5000 rpm for 5 min, and repeat the above steps.

[0084] 400 μL of Glycine / NaOH pH=9 buffer was added to resuspend the plate (OD600=6), and the plate was placed on a treated PET membrane to start the reaction.

[0085] After a period of time, the reaction was terminated, centrifuged at 12,000 rpm for 10 minutes, the supernatant was collected, and the stop solution was added. The mixture was then inactivated at 85°C for 10 minutes. The MHET absorbance of the product was measured at 240 nm. The reaction results are shown in Figure 1. In Figure 1, the wild-type strain is Pseudomonas putida carrying an empty vector.

[0086] As shown in FIG1 , the recombinant Pseudomonas putida / pMCS-INP-PETase displaying PET hydrolase on the cell surface constructed in this example exhibited catalytic activity of PET hydrolase against BHET, which was higher than that of the wild-type strain.

[0087] Example 2:

[0088] Construction and Application of Recombinant Pseudomonas putida Displaying PET Hydrolase on Cell Surface (P. putida / pMCS-PETase-AT)

[0089] Specifically, the core expression unit of the cell surface display PET hydrolase (PETase) DNA recombinant vector is constructed in the following order from 5' to 3' direction: a strong constitutive expression promoter for Pseudomonas putida (Pp_Pgro4) + a signal peptide sequence (Pp_SP) + a PET hydrolase ORF (PETaseIs, codon-optimized for Pseudomonas putida) + an anchor protein gene (estP-C, codon-optimized for Pseudomonas putida) + a Pseudomonas putida protein expression terminator (Pp_T0). The specific gene sequence is shown in SEQ ID No. 5, wherein: the Pp_Pgro4 nucleotide sequence is shown in SEQ ID No. 5, positions 1 to 244; the estP-C nucleotide sequence is shown in SEQ ID No. 2; the PETaseIs nucleotide sequence is shown in SEQ ID No. 1; and the Pp_T0 nucleotide sequence is shown in SEQ ID No. 5, positions 1826 to 1928.

[0090] Furthermore, through gene synthesis, we commissioned Sangon Biotech (Shanghai) Co., Ltd. to synthesize the cell surface-displayed PET hydrolase expression unit in pMCS-PETase-AT (with Xbal and SpeI restriction sites added upstream and downstream, respectively).

[0091] Furthermore, the pBBR1MCS plasmid (purchased from MiaoLingBio) was extracted and double-digested with Xbal and SpeI to recover the vector backbone fragment. Based on seamless cloning technology, the cell surface-displayed PET hydrolase expression unit (as shown in SEQ ID No. 5) was cloned into the pBBR1MCS linear vector after enzyme digestion to obtain the recombinant DNA plasmid pMCS-PETase-AT (pBBR1MCS+Pp_Pgro4+Pp_SP+PETaseIs+estP-C+Pp_T0), which was then transformed into Escherichia coli DH5α. After kanamycin resistance screening, colony PCR verification, and sequencing verification, the correct recombinant plasmid was obtained.

[0092] Furthermore, the pMCS-PETase-AT recombinant DNA plasmid was introduced into the host bacterium Pseudomonas putida PP1 to obtain the recombinant Pseudomonas putida P. putida / pMCS-PETase-AT displaying the PET hydrolase on the cell surface of the present invention.

[0093] Furthermore, the specific method of the above-mentioned electric shock conversion is as follows:

[0094] 1) Inoculate a single clone of Pseudomonas putida PP1 activated on an LB plate into a 4 mL / 12 mL shake tube and incubate at 30°C for 12-15 hours.

[0095] 2) Collect cells by centrifugation in a 2 mL EP tube at 13,000 × g for 2 min at room temperature;

[0096] 3) Add 1 mL of 300 mM sucrose solution to a 2 mL EP tube, resuspend the cells twice, and centrifuge at 13,000 × g for 2 minutes at room temperature to collect the cells;

[0097] 4) Resuspend the cells in 100 μL of 300 mM sucrose solution to obtain competent cells;

[0098] 5) Mix 500 ng of recombinant plasmid pMCS-SP1-PETase with 100 μL of competent cells and let stand for 5 minutes;

[0099] 6) Add the above mixture to a sterile electroporation cuvette (0.2 cm), electroporate once at 2500 V, 25 μV, and 200 Ω, add 1 mL of LB medium, transfer the cells to a 12 mL shake tube, and resuscitate at 30°C for 2 h.

[0100] 7) Spread 100 μL of the above recovery culture medium on an LB plate containing kanamycin (50 mg / L) and incubate at 30°C for 12 h.

[0101] Furthermore, a single colony of recombinant P. putida / pMCS-PETase-AT displaying PET hydrolase on its cell surface was selected from an LB plate containing 50 mg / L kanamycin and inoculated into 50 mL / 250 mL of seed culture medium. The culture was incubated at 30°C, 200 rpm for 12 h. The seed solution was centrifuged and inoculated into the fermentation medium at a final seed concentration of OD600 = 0.1. The culture was incubated at 30°C, 200 rpm for 48 h, and the bacterial broth was harvested and centrifuged at 12,000 rpm for subsequent whole-cell catalytic reactions using the recombinant strain.

[0102] Seed culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, solvent is deionized water, pH value is natural.

[0103] Fermentation medium: glycerol 5 g / L, ammonium chloride 1 g / L, KH2PO4 3 g / L, MgSO4 2 mM, Na2HPO4·12H2O 17.1 g / L, NaCl 0.5 g / L, yeast extract 2 g / L, CaCl2 0.5 g / L, VB1 40 mg / L, 1 mL / L trace element solution, solvent is deionized water, pH value is natural; trace element solution composition: ZnSO4·7H2O 2 g / L, MnCl2·2H2O 1.0 g / L, Na2B4O7 0.3 g / L, Na2MoO4·2H2O 0.2 g / L, CuSO4·7H2O, 0.2 g / L, CoCl2·6H2O 0.4 g / L, solvent is deionized water.

[0104] Whole-cell catalytic reaction of recombinant Pseudomonas putida / pMCS-INP-PETase

[0105] PET film treatment: Cut the PET film into 0.2 cm × 0.5 cm strips, rinse several times with double-distilled water, add Triton X 100 and heat-wash 1-2 times, wash with double-distilled water to remove Triton X 100 residue until no residue remains, add Na2CO3 solution and heat-wash several times, rinse several times with double-distilled water, and dry in a clean Petri dish at 60°C until used.

[0106] Take an appropriate amount of the recombinant P. putida / pMCS-PETase-AT fermentation broth, centrifuge at 3500 rpm for 5 min, add Glycine / NaOH pH = 9 buffer to resuspend, centrifuge at 5000 rpm for 5 min, and repeat the above steps.

[0107] 400 μL of Glycine / NaOH pH=9 buffer was added to resuspend the plate (OD600=6), and the plate was placed on a treated PET membrane to start the reaction.

[0108] After a period of time, the reaction was terminated and centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected, and the stop solution was added. The reaction was then inactivated at 85°C for 10 minutes. The MHET absorbance of the product was measured at 240 nm. The reaction results are shown in Figure 1.

[0109] As shown in FIG1 , the recombinant Pseudomonas putida / pMCS-PETase-AT expressing PET hydrolase on the cell surface constructed in this example exhibited catalytic activity of PET hydrolase against BHET, which was higher than that of the wild-type strain.

[0110] The present application proposes a method for displaying PET hydrolase on the surface of Pseudomonas putida. The constructed surface-display engineered bacteria can stably fix the PET hydrolase (PETase) on the outer membrane of the Pseudomonas putida cell membrane, thereby improving the ability of the PET hydrolase to be secreted and fixed on the cell surface. The bacteria can directly react with the substrate as a whole-cell catalyst without the need to disrupt the cells and purify the PET hydrolase (PETase), making the biodegradation of PET plastics more convenient and efficient. The method has the characteristics of high biocatalytic activity, realizes the application of enzymatic degradation of PET plastics, and the enzyme does not need to be separated and purified, and is simple to prepare and use, which is of great significance for achieving efficient biodegradation of PET plastics.

[0111] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0112] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. An expression unit for a PET hydrolase, characterized in that The expression unit comprises a connected constitutive expression promoter of Pseudomonas putida, an anchor protein gene, a PET hydrolase gene and a Pseudomonas putida protein expression terminator.

2. The expression unit according to claim 1, characterized in that The sequence of the PET hydrolase gene is shown in SEQ ID No. 1; And / or, the sequence of the anchoring protein gene is shown as SEQ ID No.2 or SEQ ID No.

3.

3. The expression unit according to any one of claims 1 to 2, characterized in that The nucleotide sequence of the expression unit is shown as SEQ ID No.4 or SEQ ID No.

5.

4. A recombinant vector, characterized in that: The recombinant vector contains the expression unit according to any one of claims 1 to 3.

5. The recombinant vector according to claim 4, characterized in that The original vector corresponding to the recombinant vector is selected from one of pBBR1MCS vector, pSEVA331 vector, pYMB03 vector and pJB866 vector.

6. A recombinant Pseudomonas putida, characterized in that The recombinant Pseudomonas putida contains the expression unit according to any one of claims 1 to 3; or, the recombinant Pseudomonas putida contains the recombinant vector according to any one of claims 4 to 5.

7. The recombinant Pseudomonas putida according to claim 6, characterized in that The starting host bacteria of the recombinant Pseudomonas putida is selected from one of Pseudomonas putida KT2440, Pseudomonas putida NBRC 14164, Pseudomonas putida S16, and Pseudomonas putida PP1; Furthermore, the starting host bacteria of the recombinant Pseudomonas putida is Pseudomonas putida PP1 bacteria with CCTCC NO:M 20231659.

8. A method for constructing a recombinant Pseudomonas putida, characterized in that: The steps include: An expression unit for synthesizing PET hydrolase, the expression unit comprising a connected Pseudomonas putida constitutive expression promoter, an anchor protein gene, a PET hydrolase gene and a Pseudomonas putida protein expression terminator; connecting the expression unit to a Pseudomonas putida expression vector to obtain a recombinant vector; The recombinant vector is transferred into the host Pseudomonas putida to obtain the recombinant Pseudomonas putida.

9. Use of the recombinant Pseudomonas putida obtained by the construction method of the expression unit according to any one of claims 1 to 3, or the recombinant vector according to any one of claims 4 to 5, or the recombinant Pseudomonas putida according to any one of claims 6 to 7, or the recombinant Pseudomonas putida according to claim 8 in degrading PET plastic.

10. A PET degradation agent, characterized in that: include: The recombinant Pseudomonas putida according to any one of claims 6 to 7, or the recombinant Pseudomonas putida obtained by the construction method of the recombinant Pseudomonas putida according to claim 8.

11. A PET degradation method, characterized in that: The method comprises the following steps: mixing and reacting cells with PET plastic to degrade the PET plastic, wherein the cells are the recombinant Pseudomonas putida according to any one of claims 6 to 7, or the recombinant Pseudomonas putida obtained by the construction method of the recombinant Pseudomonas putida according to claim 8.

Images